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In this paper, we show that the combination of NMR theoretical and experimental results can help to solve the molecular structure of peptides, here it is used as an example the residue Leucine-67 in Desulfovibrio vulgaris flavodoxin. We apply a computational protocol based on the leucine amino acid dipeptide, which, using calculated and experimental spin-spin coupling constants, allows us to obtain the conformation of the amino acid side chain. Calculated results show that the best agreement is obtained when three conformers around the lateral chain angle $\chi _1$ are considered or when the dynamic effect in the torsional angles is included. The population of each structure is estimated and analyzed according to the correlation between those two approaches. Independently of the approach, the estimated $\chi _1$ angle in solution is close to the staggered value of -60$^\circ $ and deviates significantly from the average x-ray angle of -90$^\circ $., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain Bacteria , are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain Archaea , none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from Methanosarcina acetivorans , an acetate-utilizing methane-producing microbe of the domain Archaea In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (-301 mV) and semiquinone/hydroquinone couples (-464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by ( i ) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and ( ii ) resilience to oxidative damage., Competing Interests: The authors declare no competing interest.

Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we have developed a research-inspired and project-based module, "From Gene to Structure and Function". This 2.5-week full-day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization. The module is centered around the flavodoxin-like protein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in-depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the students report their findings as a scientific article, with the aim to learn to present concise research results and write scientific papers. The current research-inspired project has the potential of being further developed into a more discovery-driven project and extended to include other molecular biological techniques or biochemical/biophysical characterizations. In student evaluations, this research-inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 47(3):318-332, 2019., (© 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Two flavodoxin genes from Clostridium acetobutylicum, CacFld1 (CAC0587) and CacFld2 (CAC3417), were expressed in Escherichia coli and investigated for their ability to support activity of CYP152A2, a fatty acid hydroxylase from C. acetobutylicum. E. coli flavodoxin reductase (FdR) was used as a redox partner, since flavodoxin reductase CacFdR (CAC0196) from C. acetobutylicum could not be purified in a functional form. CacFld1 was shown to accept electrons from FdR and transfer them to CYP152A2. Since H₂O₂ was generated by uncoupling at different stages of the reconstituted electron transfer chain, catalase was used as H₂O₂ scavenger in order to exclude peroxygenation by CYP152A2. The reconstituted P450 system with CacFld1 and FdR oxidized myristic acid with a K(M) of 137 μM and a k(cat) of 36 min⁻¹. Furthermore, the hydroxylase activity of CYP152A2 towards myristic acid with CacFld1 was 17-fold higher than without CacFld1. Along with CYP152A2 and a physiological flavodoxin reductase, CacFld1 is therefore likely to be involved in oxygen detoxification in C. acetobutylicum. Flavodoxin CacFld2 did not accept electrons from NADPH-reduced FdR, though it cannot be excluded as a candidate redox partner for CYP152A2 in the presence of an appropriate physiological reductase., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Flavoproteins are unique redox coenzymes, and the dynamic solvation at their function sites is critical to the understanding of their electron-transfer properties. Here, we report our complete characterization of the function-site solvation of holoflavodoxin in three redox states and of the binding-site solvation of apoflavodoxin. Using intrinsic flavin cofactor and tryptophan residue as the local optical probes with two site-specific mutations, we observed distinct ultrafast solvation dynamics at the function site in the three states and at the related recognition site of the cofactor, ranging from a few to hundreds of picoseconds. The initial ultrafast motion in 1-2.6 ps reflects the local water-network relaxation around the shallow, solvent-exposed function site. The second relaxation in 20-40 ps results from the coupled local water-protein fluctuation. The third dynamics in hundreds of picoseconds is from the intrinsic fluctuation of the loose loops flanking the cofactor at the function site. These solvation dynamics with different amplitudes well correlate with the redox states from the oxidized form, to the more rigid semiquinone and to the much looser hydroquinone. This observation of the redox control of local protein conformation plasticity and water network flexibility is significant, and such an intimate relationship is essential to the biological function of interprotein electron transfer.

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6

During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: alpha2 and beta2. The alpha subunit contains the site of nucleotide reduction, and the beta subunit contains the essential diferric-tyrosyl radical (Y*) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (alpha) and NrdB (beta), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (alpha) and NrdF (beta), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y* levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y* maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class Ib RNR. It possesses redox properties unprecedented for a flavodoxin (E(ox/sq) = -264 +/- 17 mV and E(sq/hq) = -255 +/- 17 mV) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE.

Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 66.49, c = 121.32 A. X-ray data sets have been collected to 2.17 A resolution.

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.

Escherichia coli FtsH, which belongs to the ATPases associated with diverse cellular activities (AAA) family, is an ATP-dependent and membrane-bound protease. FtsH degrades misassembled membrane proteins and a subset of cytoplasmic regulatory proteins. To elucidate the molecular mechanisms of the proteolysis, a system for precisely monitoring substrate degradation is required. We have exploited E. coli flavodoxin containing non-covalently bound flavin mononucleotide (FMN) as a model substrate for monitoring protein degradation. It was found that FtsH degrades FMN-free apo-flavodoxin but not holo-flavodoxin. However, degradation of a mutant flavodoxin carrying a substitution of Tyr94 to Asp with a lower affinity for FMN could be monitored by fluorimetry. This newly developed monitoring system will also be applicable for proteolysis by other ATP-dependent proteases.

Helicobacter pylori flavodoxin is the electronic acceptor of the pyruvate-oxidoreductase complex (POR) that catalyzes pyruvate oxidative decarboxilation. Inactivation of this metabolic route precludes bacterial survival. Because flavodoxin is not present in the human host, substances interfering electronic transport from POR might be well suited for eradication therapies against the bacterium. H. pylori flavodoxin presents a peculiar cofactor (FMN) binding site, compared to other known flavodoxins, where a conserved aromatic residue is replaced by alanine. A cavity thus appears under the cofactor that can be filled with small organic molecules. We have cloned H. pylori fldA gene, expressed the protein in Escherichia coli and characterized the purified flavodoxin. Thermal up-shift assays of flavodoxin with different concentrations of benzylamine, as well as fluorescence titration experiments indicate benzylamine binds in the pocket near the FMN binding site. It seems thus that low affinity inhibitors of H. pylori flavodoxin can be easily found that, after improvement, may give rise to leads.

Flavodoxins (Flds) are electron transfer proteins that carry a noncovalently bound flavin mononucleotide molecule (FMN) as a redox active center. A distinguishing feature of these flavoproteins is the dramatic change in the E(sq/rd) reduction potential of the FMN upon binding to the apoprotein (at pH 8.0, from -269 mV when free in solution to -438 mV in Anabaena Fld). In this study, the contribution of three neighboring FMN residues, Thr56, Asn58, and Asn97, and of three negatively charged surface residues, Glu20, Asp65, and Asp96, to modulate the redox properties of FMN upon its binding to the apoprotein has been investigated. Additionally, the role of these residues in the apoflavodoxin:FMN interaction has been analyzed. Concerning the redox potentials, the most noticeable result was obtained for the Thr56Gly mutant. In this Fld variant, the increased accessibility of FMN leads to an increase of +63 mV in the E(sq/rd) value. On the other hand, a correlation between the electrostatic environment of FMN and the E(sq/rd) has been observed. The more positive residues or the less negative residues present in the surroundings of the FMN N(1) atom, then the less negative the value for E(sq/rd). With regard to FMN binding to apoflavodoxin, breaking of hydrophobic interactions between FMN and residues 56, 58, and 97 seems to increase the K(d) values, especially in the Thr56Gly Fld. Such results suggest that the H-bond network in the FMN environment influences the FMN affinity.

Rubredoxin (Rub) from Thermotoga maritima, a 6.1-kDa red protein containing an Fe(III)-cysteine(4) center, was evaluated for its usefulness as a colored fusion tag for expression of recombinant proteins in E. coli. Here, we describe the Rub features relevant to accelerating screening for optimal high yield soluble expression conditions and automating the ensuing purification process. Spectroscopic properties and the yield of Rub fused to a typical target protein were compared to analogous GFP and Flavodoxin constructs, showing Rub absorption to be sufficient for structural genomics purposes while being produced at much higher soluble levels than GFP constructs. Based entirely on Rub absorption at 380 nm, both generic and affinity purification of crude cell lysate were performed: thus guided anion exchange purification of a Rub fusion construct as well as automated Ni-NTA purification resulted in pure protein. Rub is stable over a wide range of pH, temperature, and buffer environments, enabling robust purification protocols. Across a variety of fusion constructs, including N- and C-terminal Rub, quantitation via the Rub signal was shown to reliably correlate with analytical HPLC data obtained at 220 nm. We propose the "RubyTag" as an alternative to conventional protein fusion tags, as it combines a specific absorption signal with convenient biochemical and biological properties. Further, it allows direct on-line readout on conventional chromatography systems, holding promise for automated multi-step chromatography.

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.

Mutants of the electron-transfer protein flavodoxin from Desulfovibrio vulgaris were made by site-directed mutagenesis to investigate the role of glycine-61 in stabilizing the semiquinone of FMN by the protein and in controlling the flavin redox potentials. The spectroscopic properties, oxidation-reduction potentials, and flavin-binding properties of the mutant proteins, G61A/N/V and L, were compared with those of wild-type flavodoxin. The affinities of all of the mutant apoproteins for FMN and riboflavin were less than that of the wild-type apoprotein, and the redox potentials of the two 1-electron steps in the reduction of the complex with FMN were also affected by the mutations. Values for the dissociation constants of the complexes of the apoprotein with the semiquinone and hydroquinone forms of FMN were calculated from the redox potentials and the dissociation constant of the oxidized complex and used to derive the free energies of binding of the FMN in its three oxidation states. These showed that the semiquinone is destabilized in all of the mutants, and that the extent of destabilization tends to increase with increasing bulkiness of the side chain at residue 61. It is concluded that the hydrogen bond between the carbonyl of glycine-61 and N(5)H of FMN semiquinone in wild-type flavodoxin is either absent or severely impaired in the mutants. X-ray crystal structure analysis of the oxidized forms of the four mutant proteins shows that the protein loop that contains residue 61 is moved away from the flavin by 5-6 A. The hydrogen bond formed between the backbone nitrogen of aspartate-62 and O(4) of the dimethylisoalloxazine of the flavin in wild-type flavodoxin is absent in the mutants. Reliable structural information was not obtained for the reduced forms of the mutant proteins, but if the mutants change conformation when the flavin is reduced to the semiquinone, to facilitate hydrogen bonding between N(5)H and the carbonyl of residue 61, then the change must be different from that known to occur in wild-type flavodoxin.

The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.

Helicobacter pylori flavodoxin was purified to homogeneity from cell extracts of strain NCTC 11637. The molecular weight of the protein was estimated by gel electrophoresis to be 18 kDa. Oxidized flavodoxin showed an absorption spectrum with maxima at 378 nm and 453 nm, and it was reduced to a neutral form of flavin semiquinone by the electrons generated in the oxidation of pyruvate. This coenzyme A dependent pyruvate:flavodoxin oxidoreductase activity of H. pylori was also detected as a reduction of methyl viologen or cytochrome c by bacterial extracts. The apparent Km of pyruvate was 310 microM. Anaerobically incubated bacteria (10[9]) of strain NCTC 11637 produced acetate (96 +/- 16 nmol/h) from pyruvate concomitantly reducing metronidazole (17 +/- 5 nmol/h). In anaerobic conditions both sensitive and resistant H. pylori strains reduced metronidazole, and there was a significant positive correlation between acetate production and metronidazole activation (r = 0.77, P < 0.01, n = 11). In the presence of atmospheric oxygen, H. pylori excreted twice as much acetate but metronidazole was not activated. These results suggest that the pyruvate:flavodoxin oxidoreductase complex catalyses pyruvate oxidation in H. pylori. Electrons generated in this reaction are transferred to flavodoxin and under anaerobic conditions further to metronidazole (imidazoles) thus reducing the drug to its bactericidal form.

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum., (Copyright 1997 Academic Press.)

Electron ejection from the reduced flavin in flavodoxin from Desulfovibrio vulgaris was obtained on exposure of the protein to the third harmonic radiation (354.7 nm) generated from a pulsed Nd/YAG laser. The results indicate that the reaction is due to stepwise two-photon excitation of the reduced flavin via the excited singlet state. The absorption spectrum of the neutral flavosemiquinone radical formed in this process was obtained. This spectrum remains stable over the time of study (0.2 ms) in the pH range studied, except for a slight evolution during the first microseconds, attributed to conformational readjustments of the active site. This two-photon excitation method provides a convenient means of generating the flavosemiquinone for ultrafast kinetic studies.

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7. 5 were measured as -95 mV (Fldox/Flds) and -450 mV (Flds/Fldred) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 micromol (g protein)-1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)-1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fldox or Flds at high concentrations. The Km for Flds (80 microM) was larger than that for Fldox and for the ferredoxin isolated from W. succinogenes (15 microM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes.

Two different molecular forms of flavodoxin from the green alga Chlorella fusca have been purified to homogeneity and their properties compared. The molecular masses are 22 kDa (flavodoxin I) and 20 kDa (flavodoxin II). Western blots of axenic crude extract show the two bands. Both are single polypeptide chains and their N-terminal sequences differ but are very similar. Each form contains 1 mol of FMN/mol of apoprotein, exhibits a typical flavodoxin u.v.-visible absorption spectrum and does not contain covalently bound phosphate. The oxidation-reduction properties of the FMN in the flavodoxins differ considerably. Redox potentials of flavodoxin I at pH8 are -240 mV for the oxidized/semiquinone couple and -350 mV for the semiquinone/hydroquinone couple. Flavodoxin II gives more electronegative values: -278 mV and -458 mV respectively. Flavodoxin II fulfils better the redox requirements for photosynthetic electron transport and, as expected, it is more efficient at mediating NADP+ photoreduction in the photosynthetic electron flow. A new h.p.l.c. method for flavodoxin purification is described, which is useful for the isolation of very similar anionic proteins.

We have reported [Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. & Fukushima, S. (1992) Biosci. Biotechnol. Biochem. 56, 1780-1785] the enzymic conversion of dethiobiotin to biotin (catalyzed by the enzyme encoded by bioB) in cell-free extract of Escherichia coli which had been genetically engineered for high bioB expression. An unidentified protein(s) in addition to the bioB gene product is obligatory for this reaction. We have found that this protein was precipitated from the cell-free extract with poly(ethyleneimine), and we have purified it to homogeneity by a procedure which includes ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and Mono Q chromatography. The apparent molecular mass of the purified protein was estimated to be about 21 kDa by SDS/PAGE. The N-terminal amino acid sequence of the purified protein was identical with that of E. coli flavodoxin. We conclude that flavodoxin is required for conversion of dethiobiotin to biotin in E. coli. Studies with purified flavodoxin and the fraction containing the bioB gene product suggested that protein(s) in addition to the bioB gene product and flavodoxin is also obligatory for the reaction.

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to readily promote formation of cross-links between spinach ferredoxin-NADP+ reductase and bacterial flavodoxins. The covalent complex between ferredoxin-NADP+ reductase and the Desulfovibrio vulgaris flavodoxin had a stoichiometry of 1 mol of flavodoxin per mole of the reductase, as assessed by denaturing electrophoresis, gel filtration and spectral analysis. The reductase moiety of the cross-linked complex gained the capacity to catalyze at a high rate the electron transfer from NADPH to cytochrome c without addition of free flavodoxin in the assay. The pH optimum for this activity was shifted to the alkaline region with respect to that for the noncovalent complex. FMN, the prosthetic group of flavodoxin, is required for electron transfer from the reductase FAD to cytochrome c. Structural studies carried out on the cross-linked complex allowed the identification of the peptide regions of the proteins involved in the interaction. The CNBr peptide 61-155 of the reductase was found cross-linked to the uncleaved flavodoxin, while the cross-linked region in flavodoxin appeared to be within the tryptic peptide 37-86. Treatment of flavodoxin with the carbodiimide in the presence of glycine ethyl ester brought about the modification of a few carboxyl groups and prevented its interaction with the reductase. It can be concluded that the bacterial flavodoxin binds to the reductase in a way similar to that of the physiological substrate ferredoxin (G. Zanetti, D. Morelli, S. Ronchi, A. Negri, A. Aliverti, and B. Curti, 1988, Biochemistry 27, 3753-3759). The cross-linked complex here described represents an useful model for studying electron transfer between the two flavoproteins.

The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active. One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase. Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system. Light reduced deazaflavin can substitute for the flavodoxin system. An additional unidentified low-molecular weight component further stimulates the reaction.

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.

A flavodoxin was purified to homogeneity from the nitrogen-fixing heterocystous cyanobacterium Anabaena sphaerica grown under iron-limited conditions. The protein has a molecular mass of 21 kDa, and its spectral properties and amino-acid composition are very close to that of flavodoxins from other cyanobacteria. A. sphaerica flavodoxin supported the activities of A. sphaerica NADP reductase and Clostridium butyricum hydrogenase in reconstituted systems with illuminated plant chloroplasts as reductant. With the use of polyclonal anti-flavodoxin antiserum it was found that nitrogen-fixing cultures of A. sphaerica grown under iron-sufficient conditions contain low but significant amounts of flavodoxin (0.2-0.6 micrograms/mg crude extract protein) which increased dramatically (to 8-15 micrograms/mg crude extract protein) after the iron concentration in the medium was decreased to below 1 microM Fe. The flavodoxin content of both iron-limited and iron-sufficient. A. sphaerica was also shown to depend upon the growth phase of the (batch) cultures with a maximum at early exponential phase, coinciding with maximal in-vivo nitrogenase activity. These results suggest that A. sphaerica flavodoxin not only substitutes for ferredoxin under iron-limiting conditions, but also fulfills some specific role under iron-sufficient conditions.

The unicellular cyanobacterium Synechocystis sp PCC 6803 is capable of synthesizing two different Photosystem-I electron acceptors, ferredoxin and flavodoxin. Under normal growth conditions a [2Fe-2S] ferredoxin was recovered and purified to homogeneity. The complete amino-acid sequence of this protein was established. The isoelectric point (pI = 3.48), midpoint redox potential (Em = -0.412 V) and stability under denaturing conditions were also determined. This ferredoxin exhibits an unusual electrophoretic behavior, resulting in a very low apparent molecular mass between 2 and 3.5 kDa, even in the presence of high concentrations of urea. However, a molecular mass of 10,232 Da (apo-ferredoxin) is calculated from the sequence. Free thiol assays indicate the presence of a disulfide bridge in this protein. A small amount of ferredoxin was also found in another fraction during the purification procedure. The amino-acid sequence and properties of this minor ferredoxin were similar to those of the major ferredoxin. However, its solubility in ammonium sulfate and its reactivity with antibodies directed against spinach ferredoxin were different. Traces of flavodoxin were also recovered from the same fraction. The amount of flavodoxin was dramatically increased under iron-deficient growth conditions. An acidic isoelectric point was measured (pI = 3.76), close to that of ferredoxin. The midpoint redox potentials of flavodoxin are Em1 = -0.433 V and Em2 = -0.238 V at pH 7.8. Sequence comparison based on the 42 N-terminal amino acids indicates that Synechocystis 6803 flavodoxin most likely belongs to the long-chain class, despite an apparent molecular mass of 15 kDa determined by SDS-PAGE.

The gene coding for flavodoxin from Anabaena PCC 7119 was cloned by using the polymerase chain reaction (PCR). The gene is transcribed into a 1250-base transcript. The expression of the flavodoxin gene was analysed and found to be regulated at the transcriptional level by the availability of iron. The PCR-amplified gene was cloned into the expression vector pTrc 99b and expressed in Escherichia coli. High concentrations of flavodoxin were found (20% of total protein). The recombinant protein was purified from the cytosolic fraction of the cells and it exhibited properties identical with those of the wild-type Anabaena flavodoxin.

Proton and nitrogen-15 sequence-specific nuclear magnetic resonance assignments have been determined for recombinant oxidized flavodoxin from Anacystis nidulans (169 residues, Mr 19,048). Assignments were obtained by using 15N-1H heteronuclear three-dimensional (3D) NMR spectroscopy on a uniformly nitrogen-15 enriched sample of the protein, pH 6.6, at 30 degrees C. For 165 residues, the backbone and a large fraction of the side-chain proton resonances have been assigned. Medium- and long-range NOE's have been used to characterize the secondary structure. In solution, flavodoxin consists of a five-stranded parallel beta sheet involving residues 3-9, 31-37, 49-56, 81-89, 114-117, and 141-144. Medium-range NOE's indicate the presence of several helices. Several 15N and 1H resonances of the flavin mononucleotide (FMN) prosthetic group have been assigned. The FMN-binding site has been investigated by using polypeptide-FMN NOE's.

Two genetically distinct flavodoxins, designated AcFldA and AcFldB, were isolated from Azotobacter chroococcum (MCD1155) grown under nitrogen-fixing conditions. AcFldA and AcFldB differ in their midpoint potentials for the semiquinone-hydroquinone couple (Em -305 mV and -520 mV respectively). Only AcFldB was competent to act as an electron donor to the Mo-containing nitrogenase of A. chroococcum. The N-terminal amino acid sequence (20 residues) of AcFldB was identical with that predicted from the nifF DNA sequence of A. vinelandii OP [Bennett, Jacobsen & Dean (1988) J. Biol. Chem. 263, 1364-1369], suggesting that AcFldB is the nifF gene product of A. chroococcum (MCD1155). Direct fast reversible electrochemistry of these flavodoxins has been achieved at a polished edge-plane graphite electrode using the aminoglycoside neomycin as a promoter. The heterogeneous rates of electron transfer between the graphite electrode and AcFldA and AcFldB were determined to be 1.2 x 10(-3) cm.s-1 and 2.0 x 10(-3) cm.s-1 respectively. The natures of two minor species of flavodoxin designated AcFldC and AcFldD, which were resolved by f.p.l.c., are also discussed.

The value of a three-dimensional (3D) non-selective total correlation/nuclear Overhauser enhancement spectroscopy (TOCSY-NOESY) spectrum for making sequential resonance assignments in proteins is demonstrated using the relatively large Megasphaera elsdenii flavodoxin (molecular mass 15 kDa) in the oxidized state. An easy and concise method for the analysis of 3D-NMR spectra and a strategy for the resonance assignment of 3D-NMR protein spectra is introduced. In this context, non-selective TOCSY-NOESY is compared with selective TOCSY-NOESY and non-selective NOESY-TOCSY. Sequential assignments in various secondary structure elements of flavodoxin are made using the method of analysis introduced. NOEs not previously identified in 2D-NMR spectra due to resonance overlap are found in the 3D Clean-TOCSY-NOESY spectrum. Also additional side-chain assignments could be made.

The macroalga Porphyra umbilicalis contained two flavodoxins in approximately 5:1 ratio and differing in Mr by ca 1000. The N-terminal sequences of the isoforms were identical and there was strong immunochemical identity. Peptide mapping gave similar fragments which differed in Mr by constant amount for the two isoforms. The flavodoxins may therefore differ only at the C-terminus, possibly as a consequence of in vivo processing since inclusion of protease inhibitors during extraction had no effect on the ratio of the isoforms.

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.

The apoflavodoxin produced by precipitation of Chondrus crispus flavodoxin with trichloroacetic acid migrates as a single molecular species on non-denaturing PAGE, but at a much lower Rm than the flavoprotein. Values of s and D were significantly lower than for the flavodoxin, but their substitution in the Svedberg equation indicated the molecular mass was closely similar to that of the flavodoxin. This was confirmed by meniscus-depletion sedimentation-equilibrium studies. The Stokes radius of the apoflavodoxin was 3.65 nm, compared with 2.33 nm for the flavodoxin, and estimates of frictional coefficient ratio suggested the apoprotein was in extended conformation compared with the roughly globular shape of the flavodoxin. The Ka for FMN binding was 2.8 x 10(8)M, and the electrophoretic and physicochemical properties of the reconstituted flavoprotein were closely similar to those of the native flavodoxin. FAD, iso-FMN and thio-FMN were also bound effectively, but methyl-FMN and riboflavin were bound only weakly, if at all. The reconstituted flavoproteins were active to various extents in mediating electron transfer from NADPH to cytochrome c catalysed by flavodoxin-NADP+ oxidoreductase, the highest activity being with the thio-FMN flavodoxin.

The crystal structure of the oxidized form of a flavodoxin from an eukaryotic red alga, Chondrus crispus, has been determined by multiple isomorphous replacement and anomalous scattering methods. A model of the 173 residues and flavin mononucleotide (FMN) has been refined by a restrained least squares method to a crystallographic R-factor of 22.6% using 6236 reflections between 6.0 and 2.35 A with F greater than 3 sigma F. This molecule has a sheet consisting of five parallel beta-strands with two alpha-helices on one side of the sheet and three on the other side, and has a (beta alpha)5 structure. The molecule incorporates a substantial insertion in beta 5, as in Anacystis nidulans flavodoxin, which distinguishes these flavodoxins from the short-chain type. The isoalloxazine ring of FMN is sandwiched between the side chains of Trp-56 and Tyr-98, with its C-7 and C-8 methyl groups being exposed to solvent. The phosphate group of FMN is located at the N-terminal end of alpha 1, and forms extensive hydrogen bonds with the loop (T8-T13) between beta 1 and alpha 1 of the protein. Six of the total 11 lysine residues are clustered at the opposing face to the FMN-binding site, while about two-thirds of the total 35 acidic residues are located in the half of the molecule which includes the FMN-binding site. Such localization of charged residues produces a dipole within the molecule, which may be important in its recognition of the other proteins participating in electron transfer reactions.

Assimilatory ferredoxin-nitrite reductase (EC 1.7.7.1, ammonia: ferredoxin oxidoreductase) has been purified 5300-fold with a specific activity of 625 units/mg protein from the filamentous non-heterocystous cyanobacterium Phormidium laminosum. The enzyme was soluble and consisted of a single polypeptidic chain of 54 kDa. It catalyzed the reduction of nitrite to ammonia using ferredoxin or flavodoxin as electron donor. Methyl and benzyl viologens were also effective as electron donors but neither flavins nor NAD(P)H were. The apparent Michaelis constants for nitrite, ferredoxin and methyl viologen were 40, 22 and 215 microM, respectively. Nitrite reductase activity was inhibited effectively by cyanide and thiol reagents. The enzyme exhibited absorption maxima at 281, 391 (Soret), 570 (alpha) and 695 nm, with epsilon 391 of 4.3 x 10(4) M-1 cm-1, and an absorbance ratio A281/A391 of 1.95, suggesting the presence of siroheme as prosthetic group. These results show that this enzyme is similar to those of eukaryotic organisms.

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 degrees C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contracts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.

Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr approximately 21,000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (greater than 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.

An improved method for the purification of the holoflavodoxin from Azotobacter vinelandii was developed, which resulted in improved yields and degree of homogeneity. The purity and homogeneity of this sample were established by polyacrylamide gel electrophoresis. An apoprotein preparation procedure is outlined, which results in a homogeneous preparation of the apoflavodoxin. The homogeneity of the apoflavodoxin sample was established by polyacrylamide gel electrophoresis.